The identification of forkhead transcription factors of the Foxo sub-family as effectors of insulin action on gene expression has filled a yawning gap in our knowledge of insulin signaling, and has identified a cellular biological mechanisms linking hormone signaling to regulation of the cell's transcriptional response through sub-cellular redistribution of transcription factors. Key advances supported by this grant in the past cycle have been: i, demonstration of a key role of Foxo1 in neuropeptide processing and energy balance, linking food intake to work efficiency; ii, identification of G-protein-coupled receptor Gpr17 as a potential modulator of energy balance; iii, identification of an integrative role of hepatic Foxo in the reciprocal regulation of glucokinase vs. glucose-6-phosphatase, with important implications for the design of insulin sensitizers; iv, discovery of the metabolic functions of hepatic Notch; and v, demonstration of its functional interaction with Foxo, establishing a mechanistic link between developmental and nutrient-sensing pathways. We seek to continue our studies with the following aims: in Aim 1, we propose to investigate whether Foxo1 target Gpr17 is a transcriptional sensor of nutrient and neurotransmitter levels in Npy/AgRP neurons, linking food intake with energy expenditure and hepatic glucose production. To test the hypothesis, we generated mice lacking Gpr17 in AgRP neurons. In preliminary data, we show that these mice have metabolic changes consistent with our original hypothesis. We propose a series of studies to understand the mechanism of Gpr17 action in the CNS and in the periphery, and its functions in metabolic homeostasis. In Aim 2, we intend to study the corepressor function of Foxo1, using regulation of hepatic glucokinase as a model. The ability of Foxo to repress gene expression has been known for many years, but its effector(s) are unknown. We propose to find the hepatic Foxo1 corepressor(s). We have identified sites of Foxo1 interaction with the glucokinase promoter as well as a candidate corepressor interacting domain. We have established methods to affinity-purify Foxo1 and determine its associated proteins by mass spectrometry. Using these and other techniques in a reconstitution system of Foxo-deficient primary hepatocytes, we plan to identify and functionally characterize the Foxo1 corepressor(s).